The invention described herein may be manufactured, licensed and used by or for govermental purposes without the payment of any royalties to us thereon.
This invention relates to Directed Human Immune Globulin for the prevention and treatment of staphylococcal infections.
Over the last two decades, staphylococci have become important causes of infection in hospitalized patients. Because of their high prevalence on the skin, staphylococci are ideally situated to cause serious infections in debilitated or immunosuppressed patients. The staphylococcal species most frequently pathogenic in humans are Staphylococcus aureus (SA) and Staphylococcus epidermidis (SE). Both groups have developed resistance to multiple antibiotics making antimicrobial therapy difficult. In recent years SE has become a major cause of nosocomial infection in patients whose treatments include the placement of foreign materials such as cerebrospinal fluid shunts, vascular catheters or joint prostheses. SE is a common cause of post operative wound infections peritonitis in patients with continuous ambulatory peritoneal dialysis. Patients with impaired immunity (malignancy, bone marrow transplant) or those receiving parenteral nutrition through central venous catheter are also at high risk for developing SE sepsis (Patrick, J. Pediat., 1990).
SE has emerged as a common cause of neonatal nosocomial sepsis in premature infants. As shown by Fleer and colleagues, (Pediatr Infect Dis, 1983) SE infections frequently occur in immature babies that have received parenteral nutrition. Premature babies have impaired immunity with deficiencies in antibodies, complement and neutrophil function. Lipid infusion is now a standard ingredient of parenteral nutrition therapy in many nurseries and may further impair immunity to bacterial infection as disclosed by Fischer and colleagues (Lancet, 1980; 2:819-20). Recent studies have associated coagulase negative staphylococcal bacteria in neonates with lipid emulsion infusion (Freeman and colleagues, N. Engl. J. Med, 1990). Further studies by Fleer and colleagues (J Inf Dis, 1985) showed that neonates had low levels of opsonic antibody to SE despite the fact that the sera had clearly detectable levels of IgG antibodies to SE peptidoglycan (opsonic antibodies for staphylococcus have been considered to be directed to the peptidoglycan antigens). While these studies suggested that neonatal susceptibility to SE might be related to impaired oposonic activity, it is not clear if antibodies directed against SE are opsonic or would be capable of providing protection when given passively to neonates. Further, it is unknown whether the presence of intralipid, which further impairs phagocytosis and killing of bacteria by phagocytes, would inhibit the activity of antibody.
The opsonic activity of pooled human immunoglobulin for SE was studied by Clark and colleagues (J Med Microbiol, 19B6), and showed that complement and IgG were both critical for efficient opsonization of SE. They noted, however, that in some studies complement was not required and that contrary to the report of Fleer (1985), absorption of serum with peptidoglycan may remove the opsonic activity for SE. Further studies by Clark and Easmon (1986) showed that several lots of standard intravenous immune globulin (IVIG) had variable oposonic activity for SE. One third of the IVIG lots bad poor opsonization with complement and only 2 of 14 were opsonic without complement. Despite the fact that the IVIG lots are made from large plasma donor pools good opsonic antibody to SE was not uniformly present. Their studies focused on potential use of immunoglobulin to boost peritoneal defenses in patients receiving continuous ambulatory peritoneal dialysis and did not examine whether IVIG could be utilized for the prevention or treatment of bacterial sepsis, or the use of antibody to prevent or treat sepsis and lethal infection in immature or immunosuppressed patients and Specifically, no in vivo studies were done to test antibody to prevent or treat SE. There is no evidence therefore that the antibody would provide beneficial therapy in a setting of immaturity or impaired immunity.
The opsonic assays, that are currently used are slow and cumbersome for screening blood, plasma or immune globulin for antibodies to SE. It would be important to have a rapid antigen binding assay to screen for SE antibody, if that assay further correlated with opsonic activity in vitro and protection in vivo.
In order to determine if IgG is capable of enhancing protection against SE, a suitable animal model that is comparable to patients with SE infections is required. This is critical since neonates have low levels of complement and impaired neutrophil and macrophage function. While opsonic activity of immune globulin may be adequate under optimal conditions in vitro, protection may not occur in patients with immature or impaired immune systems. As has been demonstrated by Clark and colleagues (J Clin Pathol, 1986), most IVIG preparations were not opsonic when complement was removed. However, since SE has low virulence, suitable animal models of SE sepsis have not been available. Yoshida and collegues, (J Microbiol, 1976) reported on a virulent strain of SE that infected mature mice with 90-100% of mice dying within 24-48 hours. This model is very different from that seen in patients and may represent an unusual type of SE infection. When they analyzed 80 fresh isolates of SE from humans, they were not able to kill mice. Non-human antibody to a new SE surface polysaccharide protected the mice from the virulent SE strain. A later report by Yoshida and colleagues (J Med Microbiol, 1977) confirmed their previous observations. Passive prophylaxis with immunization induced non-human antibody showed that the IgG fraction did not protect while the IgM fraction did provide protection. Thus demonstrating in this model that IgG antibody was not protective. As noted previously herein neonates had good levels of IgG to SE, but had low levels of opsonic antibody (Fleer and colleagues, J. Infect. Dis, 1985), consistent with the findings in this study and showing that the role of IgG in protection against SE is unclear. In 1987 the report by Ichiman and colleagues (J Appl Bacteriol, 1987) extended their animal studies to include analysis of protective antibodies in human serum against their selected virulent strains of SE. Protective antibody was found in the IgA, IgM and IgG immunoglobulin fractions. These studies are in conflict with their previous data showing that IgG was not protective and fails to establish a definitive role for any of the immunoglobulin classes (IgG, IgM or IgA).
In the animal model described by Yoshida, Ichiman and colleagues mature, non-immunosuppressed mice were used and death was considered to be related to toxins not sepsis (Yoshida and colleagues, J. Microbiol, 1976). Most clinical isolates did not cause lethal infections in their model. Since quantitative blood cultures were not done, it is not known whether antibody would prevent or treat SE sepsis in immature immunosupressed patients or specifically in the presence of intralipid.
Antibody provides protection in humans against certain encapsulated bacteria such as Hemophilus influenzae and Streptococcus pneumoniae. Individuals such as young infants who are deficient in antibody are susceptible to infections with these bacteria and bacteremia and sepsis are common. When antibody to these bacteria is present it provides protection by promoting clearance of the bacteria from the blood. Immunoglobulin with antibody to H. influenzae and S. pneumoniae protects infants from sepsis with these bacteria. The article by Espersen and colleagues, (Arch Intern Med, 1987) discloses the use of an antigen binding RIA assay to analyze IgG antibody to SE in patients with uncomplicated bacteremia and those with bacteremia and endocarditis. This assay used an ultrasonic extract of SE to identify SE specific IgG (the surface antigen in this study differs from the antigen used by Yoshida and colleagues which was obtained by a different method; gentle sonic oscillation). None of the patients with uncomplicated bactermia had IgG antibodies to SE. These data would suggest that IgG is unnecessary for effective eradication of SE from the blood. In addition, 89% of bacteremic patients with endocarditis developed high levels of IgG to SE. In these patients, IgG was not protective since high levels of IgG antibody (which may have developed late) were associated with serious bacteremia and endocarditis. Based on these studies the protective role of IgG in SE sepsis and indocarditis is not established, especially in the presence of immaturity, debilitation, intralipid infusion, or immunosuppresion. In addition, the extensive review of Patrick et al. (J. Pediat., 1990) does not include immunoglobulin as a potential prophylactic or therapeutic agent for SE infections.
It has been recognized by the medical community that SE is an important pathogen in certain high risk individuals, such as patients with foreign body implants, premature neonates and immunosuppressed patients. Accordingly there is a need for a human immune globulin that would prevent or treat SE infections such as, sepsis or endocarditis and promote clearance of SE from the blood of such high risk people.
It is therefore an object of the present invention to provide a novel Directed Human Immune Globulin for preventing or treating staphylococcal infections. We have found that it is useful to screen serum (plasma) or pooled immunoglobulin for specific antibody to S. epidermidis to produce Directed Human Immune Globulin to this pathogen. This Directed Human Immune Globulin is different from standard human immume globulin preparations in that it has high levels of human anti-staphylococcal antibodies that react with surface antigens of S. epidermidis and enhance phagocytosis and killing of S. epidermidis in vitro, (opsonophagocytic bactericidal activity greater than 80%). In addition, Directed Human Immune Globulin for S. epidermidis enhances immunity in vivo and prevents lethal infection as well as enhancing clearance of S. epidermidis from the blood in conditions of immaturity and impaired immunity. This 1 is surprising since immunosuppression or immaturity would be expected to render the antibody ineffective by impairing the ability of phagocytic cells to engulf and kill the S. epidermidis. 
It is also another advantageous object of the present invention that while standard immunoglobulin pools or normal donors do not have reliable levels of opsonic antibody for S. epidermidis, Directed Human Immune Globulin when given intravenously immediately provides specific antibodies to promote phagocytosis and killing of S. epidermidis by phagocytes. A further advantages of the present invention is that by providing opsonic antibody to immature or immunosuppressed patients infected with SE, antibiotic therapy may be enhanced by improved S. epidermidis clearance from the blood or site of infection. Another advantage is that since Directed Human Immune Globulin given intravenously or intramuscularly can raise the level of antibodies in the blood of patients, Directed Human Immune Globolin could prevent S. epidermidis from causing bacteremia and local infections.
The method of producing the Directed Human Immune Globulin for S. epidermidis involves:
a) screening plasma (pools of immunoglobulin or plasma; immunoglobulin or immunoglobulin preparations) for antibodies to S. epidermidis using an in vitro antigen-binding assay: (ELISA), followed by confirmation of functional activity using an in vitro opsonophagocytic bactericidal assay (bactericidal activity greater than 80%).
b) Protective efficacy can be documented in vivo by analyzing protective activity of the Directed Human Immune Globulin using a suckling rat model of neonatal S. epidermidis sepsis (mortality and bacterial clearance). We believe that this is the first in vivo model to test antibody effectiveness in the presence of immaturity and/or intralipid induced immune suppression.
These methods could be repeated using other staphylococci such as SA instead of SE to produce Directed Human Immune Globulin for S. aureus. 
This novel Directed Human Immune Globulin for SE could be used to prevent lethal SE infections in high risk patients such as neonates and adults in intensive care units or patients with in-dwelling foreign bodies such as venous and arterial catheters or ventricular shunts. Directed Human Immune Globulin could also be used in addition to antibiotics as adjunctive therapy to enhance bacterial clearance in patients treated for SE infections.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The terms Standard Human Immunoglobulin and Directed Human Immune Globulin for S. epidermidis as used in this application are defined as follows: Standard Human immunoglobulinxe2x80x94immune human globulin that was prepared by pooling immunoglobulin from many donors, without selecting donors or screening the immunoglobulin to ensure antibody acitivity for S. Epidermidis. 
Directed Human Immune Globulin for S. epidermidisxe2x80x94Immune globulin prepared by screening for antibody to S. epidermidis (Bactericidal Activity greater than 80%), thereby providing a human immune globulin with protective levels of antibody to S. epidermidis and suitable for preventing or treating S. epidermidis infections. Bactericidal Activity-The percentage of bacteria killed with the addition of antibody, using a neutrophil mediated opsonophagocytic bactericidal assay after 2 hours of incubation at 37xc2x0 C.